Peptide c alpha-amides, methods for preparing same and uses thereof as precursors of peptide c alpha- thioesters for protein synthesis

ABSTRACT

The subject matter of the present invention is peptide C α -amides which are precursors of peptide C α -thioesters, characterized in that they comprise the radical of general formula (I) in which X, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , n and A are as defined in Claim  1 . The subject matter of the present invention is also the use of these peptide C α -amides for obtaining peptide C α -thioesters. The subject matter of the present invention is also the use of these peptide C α -amides for obtaining peptides or proteins, in particular of therapeutic interest, by direct use as a crypto-thioester partner in NCL reactions.

The present invention relates to novel peptide C^(α)-amides, theadvantage of which lies in the fact that they are direct precursors ofpeptide C^(α)-thioesters, to the methods for preparing said peptides andto the uses thereof for protein synthesis, in particular the synthesisof proteins of therapeutic interest.

Proteins are biological macromolecules which are ubiquitous in theliving world and some of which have a strong therapeutic potential. Theyare linear oligomers of α-amino acids (denoted H-Xaa-OH) linked viaamide bonds. Small proteins (less than about fifty Xaa residues) arecalled peptides. In order to be able to study their possible therapeuticapplications, it is essential to be able to readily produce them insufficient amount. The proteins required for these studies can beobtained via two routes. The biotechnological method, which involves the“recombinant DNA” technique, is often used but has many limitations.Chemical synthesis is a very advantageous additional technique for theproduction of proteins comprising up to more than two hundred α-aminoacid residues. It makes it possible to obtain proteins that aredifficult to obtain via biotechnology, such as cytotoxic proteins orproteins which are specifically modified, both on the side chains of theresidues introduced (post-translational modifications, probes,polyethylene glycol chains, etc.), but also on the peptide backbone andthe N- and C-terminal ends.

The chemical synthesis of small proteins can be carried out by SolidPhase Peptide Synthesis (SPPS) (Merrifield, B. Solid phase synthesis,Science 1986, 232, 341-347). SPPS is a technique which makes itpossible, by means of an “arm”, to attach a C-terminal amino acid whichhas suitable protective groups on a solid support, generally aninsoluble polymer called “resin”. The subsequent amino acids are addedone by one until the N-terminus is reached, by means of a succession ofchemical coupling and deprotection reactions. In this way, it ispossible to use very large excesses of reagents, thereby considerablyincreasing the reaction yields. The only purification which then becomesnecessary is abundant rinsing of the resin, carried out by simplefiltration after each reaction, the intermediate chromatographicpurification steps being eliminated. The arm is inert under all theconditions of the synthesis and allows release of the peptide insolution only during a final arm cleavage step, usually concomitant withthe deprotection of the side chains. The details of the Solid PhasePeptide Synthesis (SPPS) steps are illustrated in FIG. 1.

Two principal synthesis strategies have been developed in SPPS, namelyBoc strategy SPPS and Fmoc stragegy SPPS, which involve, respectively,α-amino acids N^(α)-protected either with a t-butyloxycarbonyl (Boc)group or with a fluorenylmethyloxycarbonyl (Fmoc) group. These two Bocand Fmoc protective groups are illustrated in FIG. 2. Boc-strategy SPPSrequires a final treatment with hydrogen fluoride (HF) which isdangerous and difficult to carry out, and is consequently increasinglyneglected. Fmoc-strategy SPPS (Fmoc SPPS) is more widely used thesedays.

SPPS is limited in terms of size of the proteins that can be routinelysynthesized (<˜50 amino acid residues). An alternative method consistsin synthesizing protein fragments via SPPS which can be condensed byvirtue of highly chemoselective reactions. These reactions are generallycarried out in a buffered aqueous medium between deprotected peptidepartners and are called chemical ligations. When the bond which resultsfrom the ligation is an amide bond, the chemical ligation is termed“native” (Dawson P. et al.; Synthesis of proteins by native chemicalligation, Science 1994, 266, 776-779). The “Native Chemical Ligation:NCL” technique most widely used is illustrated in FIG. 3. This reactionis today the reference method for the chemical synthesis of proteins.This technique involves a peptide of which the C-terminus is modifiedwith a C^(α)-thioester (see compound called (a) in FIG. 3 with R′representing a radical originating from a thiol of formula R′—SH) and apeptide bearing an N-terminal cysteine (see compound called (b)).Firstly, the sulfhydryl group of the N-terminal cysteine of the compound(b) reacts, via a trans-thioesterification reaction, with the thioesterfunction of (a). The compound (c) which results therefrom rearrangesspontaneously by acyl transfer from the sulfur to the nitrogen (S→N) viaa five-membered cyclic transition state, which is highlythermodynamically favored. A peptide (d) which has an amide bond at thejunction between the two fragments is thus obtained.

Contrary to the synthesis of peptide C^(α)-acids and C^(α)-amides,peptide C^(α)-thioesters are particularly difficult to synthesize byFmoc SPPS owing to the non-compatibility of the thioester function(—CO—S—) with the piperidine used to deprotect the Fmoc group.Piperidine is in fact a nucleophilic amine which rapidly reacts withthioester functions to cleave the carbon-sulfur bond and to give thecorresponding amide and thiol. The vast majority of the contemporarystrategies for the synthesis of peptide thioesters by Fmoc SPPS areindirect methods: the thioester function is introduced only after havingcarried out the complete extension of the peptide.

Among the various existing methods for synthesizing peptideC^(α)-thioesters by Fmoc SPPS (FIG. 4-2, compound (a)), some involvepeptide C^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides (FIG. 4-1, compound (e)with R=alkyl and R″ representing a group comprising a function havingmade it possible, during the first steps of the synthesis of (e), tograft a precursor of (e) to an insoluble polymer by means of an arm) orpeptide C^(α)-N-aryl-N-(β-mercaptoalkyl)-amides (FIG. 4-1, compound (e)with R=aryl and R″ as previously defined).

Such peptide C^(α)-β-mercapto-amides (e) are generally spontaneously inequilibrium with a C^(α)-β-amino-thioester form (f) via anintramolecular acyl transfer from the nitrogen to the sulfur (N→S)detailed in FIG. 4-1. The equilibrium is shifted toward the amide form(e) under neutral or basic conditions, whereas the thioester form (f)predominates in an acidic medium. Reaction with an excess of thiol(R′SH), at an acidic pH, of compounds of (e) or (f) type makes itpossible to gradually shift this equilibrium and to obtain the peptideC^(α)-thioester (a) devoid of β-amine function (compound (a)).

It has been shown in several recent examples that some of these peptideC^(α)-β-mercapto-amides (e) can be used directly, in the same way as thepeptide C^(α)-thioesters (f), under NCL conditions, i.e. in the presenceof a peptide which has an N-terminal cysteine of type (b) and of anexcess of thiol R′SH, in a medium buffered at a pH close to neutrality.The principle is, a priori, that the N→S acyl transfer and thetrans-thioesterification take place in situ at the time of the ligation(FIG. 4-3). The peptide C^(α)-β-mercapto-amides (e) which have thisproperty are called “crypto-thioesters”. Only a few examples have beendescribed to date (1-Ollivier N. et al.; Bis(2-sulfurnylethyl)aminonative peptide ligation, Org. Lett. 2010, 12, 5238-5241; 2-Sato K. etal.; Nsulfurnylethylanilide peptide as a crypto-thioester peptide,ChemBioChem 2011, 12, 1840-1844; 3-Hou W. et al.; PeptidylN,N-bis(2-mercaptoethyl)-amides as thioester precursors for nativechemical ligation. Organic Letters 2011, 13, 386-389).

The synthesis of peptide C^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides orC^(α)-N-aryl-N-(β-mercaptoalkyl)-amides (see FIG. 5-1, compound (e) withR=alkyl or aryl) is carried out by Fmoc SPPS while masking the thiolfunction of the compound (h) with a protective group (Protec), theconsequence of which is to prevent rearrangement in a thioester form oftype (f) (FIG. 4-3). The peptide remains in its amide form, and istherefore stable throughout the elongation.

The main drawback of this approach of the synthesis of thioesters or ofcrypto-thioesters lies in the fact that all the syntheses described todate have in common an extremely difficult first step of N-acylation(FIG. 5-1). Indeed, because of the steric hindrance around the nitrogenatom, the reaction kinetics are generally very slow, which can result inincomplete N-acylation, unwanted by-products and low yields. In manycases, this approach is limited in practice to peptide C^(α)-thioesterswhich have a glycine residue in the C-terminal position.

There still remains therefore the need to synthesize peptideC^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides orC^(α)-N-aryl-N-(β-mercaptoalkyl)-amides by means of a simple andefficient method. Indeed, a simple and efficient preparation of suchpeptide C^(α)-amides allows in particular a simple and efficientpreparation of peptide C^(α)-thioesters, since these peptideC^(α)-amides are direct precursors of peptide C^(α)-thioesters.

In addition, such peptide C^(α)-amides may also have crypto-thioesterproperties, and may be used directly in an NCL reaction without it beingnecessary to isolate the thioester form beforehand.

Advantageously according to the invention, the N-acylation of thesecondary amine of type (h) (FIG. 5-1) is advantageously accelerated, inparticular by virtue of an entirely original radical R which allowsintramolecular assistance during the N-acylation.

The principle of such an assistance for the acylation of a secondaryamine has been described in the literature, but never applied to peptideC^(α)-β-mercapto-amide precursors of C^(α)-thioesters. By way ofexample, mention may be made of the assistance of the acylation of asecondary amine of a peptide by an N-(2-hydroxybenzyl) function (JohnsonT. et al.; A reversible protecting group for the amide bond in peptides.Use in the synthesis of “difficult sequences”, J. Chem. Soc. Chem.Commun., 1993, 369-374).

This assistance is illustrated in FIG. 5-2. The N-acylation of thesecondary amine (j) is much faster than that of the secondary amine (m),even though the steric hindrance around the nitrogen atom (principalfactor determining the kinetics of a conventional N-acylation reaction)of each of these two compounds is comparable. This great difference inreactivity is due to the presence of an N-(2-hydroxybenzyl) group in (j)but not in (m). The hydroxyl function of this group can be O-acylatedunder N-acylation conditions, to give an intermediate (k). Contrary tothe secondary amine function, the hydroxyl function is barely hinderedat all, and this O-acylation reaction is rapid. The intermediate (k)will rapidly undergo acyl transfer from the oxygen to the nitrogen (O→N)so as to give the amide (l) via a six-membered cyclic transition statewhich is highly thermodynamically favored. This N-acylation byintramolecular acyl transfer resulting in (l) is hardly at all sensitiveto the steric hindrance around the nitrogen atom, contrary to theintermolecular version resulting in (n), thereby explaining the muchfaster N-acylation of (j) compared with (m). A similar assistance hasalso been mentioned during the acylation of peptides which have an aminefunction that has a group of γ-(2-azaheterocycle) type (Zhang L. et al.;Orthogonal coupling of unprotected peptide segments through histidylamino terminus, Tetrahedron Lett. 1997, 38, 3-6).

One of the objectives of the present invention is to provide a methodfor the synthesis of peptide C^(α)-N-alkyl-N-(β-mercaptoalkyl)-amideprecursors of peptide C^(α)-thioesters which is at the same time simpleto carry out, rapid and inexpensive, and which does not generateunwanted by-products.

Advantageously according to the invention, some of the groups present onthe original radicals R as mentioned previously may also allowintramolecular assistance of the N→S acyl transfer and/or of thetrans-thioesterification, and thus increase the ability of the peptideC^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides of the invention to behave likecrypto-thioesters. This assistance may result, for example, from theformation of hydrogen bonds, dipole interactions, π-π interactions, oracid-base catalysis.

Another objective of the invention is to provide a method for thesynthesis of original peptide C^(α)-N-alkyl-N-(β-mercaptoalkyl)-amideswhich have crypto-thioester properties in order to be able to bedirectly used as a partner in NCL reactions.

In the present application, the peptideC^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides will be more simply referred toas peptide C^(α)-amides. The peptide C^(α)-amides of the invention coverboth said peptide C^(α)-N-alkyl-N-(β-mercaptoalkyl)-amides and alsopeptide C^(α)-N-alkyl-N-(γ-mercaptoalkyl)-amides,C^(α)-N-alkyl-N-(β-selenoalkyl)-amides andC^(α)-N-alkyl-N-(γ-selenoalkyl)-amides, said peptide C^(α)-amides of theinvention being precursors of peptide C^(α)-thioesters, according to apresumed mechanism of acyl transfer from the sulfur or the selenium tothe nitrogen.

The peptide C^(α)-amides of the invention may also be referred to as“crypto-thioester” peptides owing to the possibility of using themdirectly in NCL reactions, without it being necessary to prepare thethioester form beforehand.

A subject of the present invention is thus a peptide C^(α)-amideprecursor of a peptide C^(α)-thioester, characterized in that itcomprises the radical of general formula (I):

-   -   in which    -   X represents a sulfur or selenium atom,    -   R₁ represents a hydrogen atom or a protective group for the        sulfur or for the selenium which is compatible with conditions        of elongation by Fmoc SPPS,    -   m represents an integer equal to 0 or 1,    -   one of R₂, R₃, R₄, R₅, R₆ or R₇ represents the radical —B—C-D in        which:        -   D represents a hydrogen atom or a solid support suitable for            solid phase peptide synthesis (SPPS),        -   C is absent or represents an arm that can be used for SPPS,        -   B represents a divalent radical comprising a heteroatom,    -   the others of said R₂, R₃, R₄, R₅, R₆ or R₇ which do not        represent —B—C-D, then represent, independently of one another,        a hydrogen atom, or an alkyl radical having from 1 to 10 carbon        atoms, and preferably a methyl radical (—CH₃) or a phenyl        radical (—C₆H₅), on the condition that at most two of said        radicals R₂, R₃, R₄, R₅, R₆ or R₇ represent at the same time a        phenyl,    -   R₈ and R₉ represent, independently of one another, an alkyl        radical having from 1 to 10 carbon atoms, and preferably a        methyl radical, or a hydrogen atom,    -   n represents an integer ranging from 0 to 4, preferably from 0        to 1,    -   A representing an aryl or heteroaryl radical chosen from the        group comprising        -   the radical of formula:

-   -   -   in which        -   R₁₀ represents an alkyl radical having from 1 to 10 carbon            atoms, and preferably a methyl,        -   R₁₁ and R₁₂ represent, independently of one another, a            hydrogen atom, a halogen atom chosen from the group            comprising Cl, Br, I and F, a CN radical, an —NO₂ radical, a            —CF₃ radical, a phenyl radical (—C₆H₅), a —CONH₂ radical, an            R₁₀ radical, an —OR₁₀) radical, an —SR₁₀ radical, an            —N(R₁₀)₂ radical, a —COOR₁₀ radical, a —CONHR₁₀ radical or a            —CON(R₁₀)₂ radical, R₁₀ being as previously defined,        -   the radical of formula:

-   -   -   in which        -   at least one of X₁, X₂, X₃, X₄ and X₅ represents a            nitrogen (N) atom, a C—OH radical or a C—SH radical, the            others of said X₁, X₂, X₃, X₄ or X₅ which do not represent            N, C—OH or C—SH, then representing, independently of one            another, a C—R₁₁ radical with R₁₁ as previously defined,        -   the radical of formula:

-   -   -   in which        -   at least one of X₁, X₂, X₃ and Y₄ represents a nitrogen (N)            atom, a C—OH radical or a C—SH radical, the others of said            X₁, X₂ or X₃ which do not represent N, C—OH or C—SH, then            representing, independently of one another, a C—R₁₁ radical            with R₁₁ as previously defined,        -   Y₁, Y₂, Y₃ and Y₄ (on condition that Y₄ does not represent            C—OH or C—SH) represent, independently of one another,            depending on whether they are linked via a single or double            bond, a nitrogen (N) atom or an NR₁₀ group with R₁₀ as            previously defined, a CH or CH₂ group, a C—R₁₁ or CHR₁₁ or            CR₁₁R₁₂ group with R₁₁ and R₁₂ as previously defined, a            carbonyl (C═O), an oxygen (O) atom or a sulfur (S) atom,            with the condition that at most two of said Y₁, Y₂, Y₃ and            Y₄ represent at the same time an oxygen atom or a sulfur            atom, one of said Y₁, Y₂, Y₃ or Y₄ possibly being absent, so            as to form a 5-membered ring.

The term “alkyl” is intended to mean a hydrocarbyl radical having 1 to10 carbon atoms, corresponding to the general formula C₁₁H₂₊₁ where n isgreater than or equal to 1. The alkyl groups may be linear or branchedand may be substituted with the groups indicated in the presentapplication.

The term “aryl” is intended to mean an aromatic polyunsaturatedhydrocarbyl group having a single ring (for instance phenyl) or severalfused rings (for instance naphthyl) or several rings connected via acovalent bond (for instance biphenyl), which typically contain 5 to 12carbon atoms, preferentially 6 or 12, and where at least one ring isaromatic. The aromatic ring may optionally comprise one to twoadditional fused rings (i.e. cycloalkyl, heterocycloalkyl orheteroaryl). The term “aryl” also comprises the partially hydrogenatedderivatives of carbocyclic systems described above. The aromatic ringmay optionally comprise one to five substituents (other than H) of alkyl(preferentially methyl), —F, —Cl, —Br, —I, —CF₃, —OMe, —SMe, —N(Me)₂,—COOH, —SO₃H, —CH₂N(Me)₂, —CH₂COOH, —CH₂SO₃H, —CH₂CH₂N(Me)₂,—CH₂CH₂COOH, —CH₂CH₂SO₃H, —OCH₂COOH, —OCH₂SO₃H, —OCH₂CH₂N(Me)₂,—OCH₂CH₂COOH or —OCH₂CH₂SO₃H type.

The term “heteroaryl” is intended to mean a ring or several fused ringsor rings connected via a covalent bond, comprising 3 to 17 carbon atoms,preferentially 3 to 11 carbon atoms, where at least one of the rings isaromatic and where at least one or more carbon atoms are replaced withoxygen, nitrogen and/or sulfur. The term “heteroaryl” also comprisessystems described above having a fused aryl, cycloalkyl, heteroaryl orheterocycloalkyl group or one to five alkyl, preferentially methyl,substituents. The term “heteroaryl” also comprises systems describedabove comprising one to four substituents (other than H) of alkyl(preferentially methyl), —F, —Cl, —Br, —I, —CF₃, —OMe, —SMe, —N(Me)₂,—COOH, —SO₃H, —CH₂N(Me)₂, —CH₂COOH, —CH₂SO₃H, —CH₂CH₂N(Me)₂,—CH₂CH₂COOH, —CH₂CH₂SO₃H, —OCH₂COOH, —OCH₂SO₃H, —OCH₂CH₂N(Me)₂,—OCH₂CH₂COOH or —OCH₂CH₂SO₃H type.

The term “cycloalkyl” is intended to mean a saturated or unsaturated,cyclic monovalent hydrocarbyl having one or two rings and comprising 3to 10 carbon atoms.

The term “heterocycloalkyl” is intended to mean a cycloalkyl in which atleast one carbon atom is replaced with an oxygen, nitrogen and/or sulfuratom.

According to one advantageous embodiment of the invention, the divalentradical B present in the radical (I), represents:

-   -   in which    -   E is absent or represents the -(Xaa)_(i)- group in which:    -   i represents an integer ranging from 1 to 20, and preferably        from 1 to 5,    -   each Xaa represents, independently of one another, an amino acid        residue, and    -   when i is greater than or equal to 2, each of said Xaa is        connected to its neighboring Xaa via a peptide bond,    -   or, in the case where m=1, and R₄ or R₅=—B—C-D, then the        divalent radical B preferably represents:

-   -   in which:    -   j represents an integer ranging from 0 to 10, preferably from 0        to 3, and E is as previously defined.

The possible reactive functions of the side chains of said amino acidresidues may be free, i.e. unprotected, or conversely protected withprotective groups normally used by those skilled in the art during SPPS.

According to another advantageous embodiment of the invention, in theradical (I), C represents an arm that can be used for Fmoc SPPS, andpreferably an acid-labile arm chosen from the group comprising a Rink(4-[(2,4-dimethoxyphenyl)methyl]phenoxyacetyl) arm, a Wang(4-alkoxybenzyl) arm, a Sieber (xanthen-3-yloxyalkyl) arm, a PAL(4-2,5-dimethoxyalkoxybenzyl) arm, a 2-chlorotrityl arm, a PAM(phenylacetamidomethyl) arm, a SASRIN (2-methoxy-4-alkoxybenzyl) arm oran MBHA (4-methyl)benzhydryl arm.

According to another advantageous embodiment of the invention, in theradical (I), D represents a solid support suitable for Fmoc SPPS and ispreferably chosen from the group comprising a resin sold under the namePega®, a resin sold under the name Chemmatrix™, a polyacrylamide resin,a polystyrene resin or a polystyrenepolyethylene glycol (PEG) mixedresin.

By way of example of a polystyrenepolyethylene glycol (PEG) mixed resin,mention may be made of those sold under the name tentagel or argogel.

According to one advantageous embodiment of the invention, in theradical (I), n is an integer equal to 0, and the radical A has theformula:

-   -   in which X₁ represents C—OH, and each of X₂, X₃, X₄ and X₅ is as        previously defined.

Preferably, each of X₂, X₃ or X₅ represents CH and X₄ represents C—R₁₁with R₁₁ as previously defined.

Even more preferably, X₄ represents CH, C-OMe or C—NO₂.

Thus, a preferred radical A is the one represented by one of thefollowing formulae:

According to another advantageous embodiment of the invention, in theradical (I), n is an integer equal to 0, and the radical A has theformula:

-   -   in which X₁ represents a nitrogen (N) atom and each of said X₂,        X₃, X₄ and X₅ is as previously defined. Preferably, each of X₂,        X₃ or X₅ represents CH and X₄ represents CH or C—R₁₁ with R₁₁        representing OCH₃ or N(CH₃)₂.

Thus, another preferred radical A is the one represented by one of thefollowing formulae:

According to another advantageous embodiment of the invention, in theradical (I), n is an integer equal to 1, and the radical A has theformula:

-   -   in which X₁ represents a nitrogen (N) atom and each of said X₂,        X₃, X₄ and X₅ is as previously defined. Preferably, each of X₂,        X₃, X₄ and X₅ represents CH.

Thus, another preferred radical A with n equal to 1 is the onerepresented by the following formula:

According to another advantageous embodiment of the invention, in theradical (I), n is an integer equal to 0, and the radical A has theformula:

-   -   in which X₁ or Y₄ represents C—OH or N, the other of said X₁ or        Y₄ which does not represent C—OH or N and also each of said X₂,        X₃, Y₁, Y₂ and Y₃ are as previously defined.

According to another advantageous embodiment of the invention, in theradical (I), n is an integer equal to 0, and the radical A has theformula:

-   -   in which X₁ or X₂ represents C—OH or N, the other of said X₁ or        X₂ which does not represent C—OH or N and also each of said X₃,        Y₁, Y₂, Y₃ and Y₄ are as previously defined.

According to one preferred embodiment of the invention, the peptideC^(α)-amide is characterized in that, in the radical (I):

-   -   m represents 0 or 1,    -   each of said R₃, R₄, R₅, R₆ or R₇ represents H,    -   R₂ represents —B—C-D in which B represents:

-   -   and C, D and E are as previously defined.

According to one preferred embodiment of the invention, in the radical(I), m is equal to 0 and X represents a sulfur (S) atom.

When R₁ is a protective group for sulfur, then said group can be chosenfrom the group comprising trityl (Trt), methyltrityl (Mtt),methoxytrityl (Mmt), xanthenyl (Xan), tri-methoxybenzyl (Tmob),acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), benzamidomethyl(Bam), allyloxycarbonylaminomethyl (Allocam), phthalimidomethyl (Pim),2-(trimethylsilyl)ethyl, t-butyl (tBu), 2,4,6-trimethylbenzyl,quinolinylmethyl (Qm), diphenyl-4-pyridylmethyl, 1-adamantyl,benzyloxymethyl (BOM), 2-tetrahydropyranyl (Thp), benzylthiomethyl,ethylsulfenyl (SEt), t-butylsulfenyl (StBu), phenylsulfenyl (SPh) and2,4-dinitrophenyl.

More particularly, according to one advantageous embodiment of theinvention, when R₁ is a protective group for sulfur which can be cleavedby treatment with trifluoroacetic acid (TFA), then said group ispreferably chosen from the group comprising trityl (Trt), methyltrityl(Mtt), methoxytrityl (Mmt), xanthenyl (Xan) and tri-methoxybenzyl(Tmob).

According to another advantageous embodiment of the invention, when R₁is a protective group for sulfur which is stable with respect totreatment with TFA and stable under NCL conditions, then said group ispreferably chosen from the group comprising acetamidomethyl (Acm),trimethylacetamidomethyl (Tacm), benzamidomethyl (Bam),allyloxycarbonylaminomethyl (Allocam), phthalimidomethyl (Pim),2-(trimethylsilyl)ethyl, t-butyl (tBu), 2,4,6-trimethylbenzyl,quinolinylmethyl (Qm), diphenyl-4-pyridylmethyl, 1-adamantyl,benzyloxymethyl (BOM), 2-tetrahydropyranyl (Thp) and benzylthiomethyl.

According to yet another advantageous embodiment of the invention, whenR₁ is a protective group for sulfur which is stable with respect totreatment with TFA, but labile under NCL conditions, then said group ispreferably chosen from the group comprising ethylsulfenyl (SEt),t-butylsulfenyl (StBu), phenylsulfenyl (SPh) and 2,4-dinitrophenyl.

According to yet another advantageous embodiment, R₁ is a group which islabile in an NCL native chemical ligation reaction and is preferablychosen from the group containing ethylsulfenyl (SEt), t-butylsulfenyl(StBu) and phenylsulfenyl (SPh) groups.

According to one advantageous embodiment of the invention, X representsa selenium (Se) atom, and R₁ is a protective group for selenium and ispreferably chosen from the group comprising 4-methoxybenzyl (Mob),4-methylbenzyl (MeBz1) or benzyl (Bzl).

By way of example of a radical (I) according to the invention, mentionmay be made of one of those chosen from the group comprising:

-   -   in which:    -   B represents

and E, C, D and R₁ are as previously defined.

The peptide C^(α)-amide which is the subject of the invention is moreparticularly characterized in that it has general formula (II):

-   -   in which:        -   Peptide1 represents the R₁₈-(Xaa)_(k)- group in which:        -   k is an integer ranging from 1 to 100, preferably from 4 to            60, and even more preferentially from 8 to 40,        -   Xaa represents, independently of one another, an amino acid            residue originating from an amino acid of formula H-Xaa-OH,            and when k is greater than or equal to 2, each of said Xaa            is connected to its neighboring Xaa via a peptide bond,        -   R₁₈ is a hydrogen atom or a substituent of the N-terminal            end included in the Xaa residue,        -   X, m, n, A and R₁ to R₉ being as previously defined.

Preferably, in the divalent radical B as defined above, E is absent orrepresents -(Xaa)_(i)- which is chosen so as to increase the aqueoussolubility of the Peptide 1.

The possible reactive functions of the side chains of the amino acidresidues -(Xaa)_(k) may be free, i.e. unprotected, or converselyprotected with protective groups normally used by those skilled in theart during SPPS.

According to a first advantageous embodiment of the invention, theprocess for preparing the compound of formula (Ia) (said compound (Ia)forming the radical (I) within the peptide C^(α)-amide of the invention)is characterized in that it comprises:

-   -   a step of grafting, onto a solid support of formula:

H-E-O—C-D′, H-E-NH—C-D′ or Cl—C-D′

in which E and C are as previously defined, D′ represents a resinsuitable for SPPS and H, O, NH and Cl correspond to the chemical symbolsnormally used, a compound of general formula (III):

-   -   in which:    -   R₁₃ represents a protective group for the amine function, and        preferably an Fmoc group,    -   m, X and R₁ are as previously defined,    -   one of R′₂, R′₃, R′₄, R′₅, R′₆ or R′₇ representing a reactive        group comprising a reactive chemical function preferably chosen        from the group comprising —COOH, —NH₂ and —OH, and making it        possible to graft said compound (III) onto the solid support as        mentioned above according to a method known to those skilled in        the art for Fmoc SPPS,    -   the others of said R′₂, R′₃, R′₄, R′₅, R′₆ or R′₇ which do not        represent said reactive group have the same meaning as said R₂,        R₃, R₄, R₅, R₆ or R₇ as previously defined and do not represent        —B—C-D,    -   in order to obtain a compound of general formula (IV):

-   -   in which:    -   m, X, R₁ and R₁₃ are as previously defined,    -   one of R″₂, R″₃, R″₄, R″₅, R″₆ or R″₇ represents —B—C-D′ in        which B, C and D′ are as previously defined,    -   the others of said R″₂, R″₃, R″₄, R″₅, R″₆ or R″₇ which do not        represent the radical —B—C-D′ have the same meaning as said R₂,        R₃, R₄, R₅, R₆ or R₇ as previously defined and do not represent        the radical —B—C-D,    -   said grafting step being followed by a step of cleaving the        protective group R₁₃ of the compound (IV) in order to obtain a        compound of general formula (IVa):

-   -   in which:    -   m, X, R₁, R″₂, R″₃, R″₄, R″₅, R″₆ and R″₇ are as previously        defined,    -   said cleaving step being followed by a step of mono-N-alkylation        of the compound (IVa) in order to obtain the compound of general        formula (V):

-   -   in which X, R₁, m, R″₂, R″₃, R″₄, R″₅, R″₆, R″₇, n, R₈, R₉ and A        are as previously defined,    -   the compound (V) being a direct precursor of the compound of        general formula (Ia):

-   -   in which:    -   R₂₀═H or R₁₃ as previously defined,    -   X, R₁, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as        previously defined,    -   the compound (Ia) being obtained from the compound (V) according        to one of the methods known to those skilled in the art.

The compound (Ia) forms the radical (I) within the peptide C^(α)-amideof the invention (see, for example, the compound of general formula(II)).

According to one advantageous embodiment of the process for preparingthe compound (Ia) of the invention, the step of mono-N-alkylation of thecompound (IVa) comprises a reaction for reductive amination of saidcompound (IVa) using an aldehyde of general formula (VI):

-   -   in which A, n, R₈ and R₉ are as previously defined,    -   in order to form an imine which is reduced so as to form the        compound (V) as defined above.

The reduction may, for example, be carried out using a borohydridechosen from the group comprising sodium cyanoborohydride (NaBH₃CN),sodium triacetoxyborohydride (NaBH(OAc)₃) or sodium borohydride (NaBH₄).

According to another advantageous embodiment of the process forpreparing the compound (Ia) of the invention, the step ofmono-N-alkylation of the compound (IVa) comprises:

-   -   a prior step of sulfonylation of the primary amine function of        the compound (IVa), in order to obtain the compound of formula        (Nb):

-   -   in which R₁₉ represents an arylsulfonyl group chosen from the        group comprising 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl        and 2,4-dinitrobenzenesulfonyl,    -   m, X, R₁, R″₂, R″₃, R″₄, R″₅, R″₆ and R″₇ are as previously        defined,    -   said sulfonylation step being followed by a step of        monoalkylation by nucleophilic N-substitution between the        compound (Wb) and a compound of general formula (VII):

-   -   in which Z represents a leaving group chosen from the group        comprising Cl, Br, I or a sulfonate, it being possible for said        sulfonate to be, for example, an “O-mesylate” (—OMs) represented        by MeSO₂—O—, an “O-tosylate” (—OTs) represented by        p-Me-C₆H₄—SO₂—O— or an “O-triflate” (—OTf) represented by        CF₃SO₂—O—,    -   A, n, R₈ and R₉ are as previously defined,    -   so as to obtain the compound of formula (Va):

-   -   in which m, X, R1, R″₂, R″₃, R″₄, R″₅, R″₆, R″₇, A, n, R₈ and R₉        are as previously defined,    -   said monoalkylation step being followed by a step of cleaving        the sulfonyl group R₁₉ of the compound (Va) in order to obtain        the compound of general formula (V), the compound (V) being a        direct precursor of the compound of general formula (Ia), said        compound (Ia) forming the radical (I) within the peptide        C^(α)-amide of the invention.

According to a second advantageous embodiment of the invention, theprocess for preparing the compound (Ia) is characterized in that itcomprises:

-   -   a step of mono-N-alkylation of a compound of general formula        (Ma):

-   -   in which X, R₁ and m are as previously defined,    -   one of R′″₂, R′″₃, R′″₄, R′″₅, R′″₆ or R′″₇ represents a        reactive group comprising a reactive chemical function        optionally protected with a protective group, said function        being preferably chosen from the group comprising —COOH, —NH₂        and —OH, and making it possible to graft said compound (Ma) onto        the solid support as mentioned above according to a method known        to those skilled in the art for Fmoc SPPS, said optional        protective group being preferably chosen from a group comprising        tert-butyl, tert-butyloxycarbonyl, phenacyl, benzyl,        benzyloxycarbonyl, methyl, ethyl, trityl and allyl,    -   the others of said R′″₂, R′″₃, R′″₄, R′″₅, R′″₆ or R′″₇ which do        not represent said reactive group have the same meaning as said        R₂, R₃, R₄, R₅, R₆ or R₇ as previously defined and which do not        represent —B—C-D,    -   in order to obtain a compound of general formula (VIII):

-   -   in which X, R₁, m, R′″₂, R′″₃, R′″₄, R′″₅, R′″₆, R′″₇, n, R₈, R₉        and A are as previously defined,    -   a step of N-acylation or of protection of the secondary amine        function of the compound of formula (VIII) in order to obtain a        compound of general formula (VIIIa):

-   -   in which R₁₄═R₁₃ as previously defined or R₁₄ represents a        protected aminoacyl residue of formula R₁₃—Xaa- with Xaa        representing an amino acid residue, the possible reactive        functions of the side chain of the amino acid residue Xaa being        protected with protective groups normally used by those skilled        in the art during SPPS,        -   an optional step of deprotection of the reactive function            included in one of said R′″, so as to give a compound of            general formula (VIIIb)

-   -   -   a step of grafting the compound (VIIIb) onto a solid support            of formula:

H-E-O—C-D′, H-E-NH—C-D′ or Cl—C-D

-   -   in which E, C and D′ are as previously defined and H, O, NH and        Cl correspond to the chemical symbols normally used, in order to        obtain the compound of formula (Vb):

-   -   in which    -   X, R₁, m, R″₂, R″₃, R″₄, R″₅, R″₆, R″₇, R₁₄, n, R₈, R₉ and A are        as previously defined,    -   in the case where R₁₄═R₁₃, said grafting step being followed by        a step of cleaving the R₁₄ group of the compound (Vb) in order        to obtain a compound of general formula (V) as previously        defined, said compound (V) being a direct precursor of a        compound of formula (Ia) as previously defined,    -   in the case where R₁₄═R₁₃-Xaa-, said grafting step being        followed by a step of cleaving the R₁₃ group which is part of        the R₁₄ group of the compound (Vb) in order to obtain a compound        of general formula (Vc),

-   -   said compound (Vc) being the precursor of a peptide C^(α)-amide        of the invention comprising a radical (I), by elongation of the        peptide chain by SPPS.

A subject of the invention is also a compound characterized in that ithas general formula (Ia):

-   -   in which:    -   X, R₁, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as        previously defined and R₂₀ represents a hydrogen or R₁₄.

A subject of the invention is also a process for preparing a peptideC^(α)-amide of formula (II) as defined above by Fmoc SPPS, characterizedin that it comprises a step of elongation, by Fmoc SPPS, of the compound(Ia) as previously defined, said elongation step making it possible toadd Peptide1 as previously defined.

Advantageously according to the invention, the attachment of the firstamino acid to the compound (Ia) by N-acylation (namely the addition ofan amino acid to the nitrogen of the compound (Ia)), may be carried outvery easily by virtue of the actual structure of the compound (Ia).

Indeed, the use of the compound of the invention (Ia) is extremelyadvantageous, compared, for example, with the compound (h) of the priorart defined in FIG. 5-1, since it is the original structure of saidcompound (Ia) which makes it possible to facilitate this step ofN-acylation of the secondary amine.

A subject of the present invention is also a process for preparing apeptide C^(α)-thioester of general formula (IX):

Peptide1-S—R′  (IX)

-   -   in which:    -   R′— represents a radical originating from a thiol of formula        R′—SH, and Peptide1 is as previously defined,    -   characterized in that it comprises a reaction for        thioesterification between:        -   the peptide C^(α)-amide of formula (II) as previously            defined in which R₁═H, and        -   a thiol of formula R′—SH,    -   in order to obtain:        -   the peptide C^(α)-thioester of formula (IX) as defined            above, and        -   the compound of formula (Ib):

-   -   in which:    -   X, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as previously        defined.

The compound (Ib) corresponds to the compound of general formula (Ia) inwhich R₁ and R₂₀ each represent a hydrogen atom.

The R′— radical originating from the thiol of formula R′—SH mayrepresent any radical which makes it possible to form a thiol (R′—SH)when it is bonded to the SH function of the thiol.

The R′— radical may, for example, represent an alkyl, aryl, arylalkyl,cycloalkyl, heterocycloalkyl or heteroaryl group as defined above, itbeing possible for each of said groups to also comprise one or moreconventional substituents chosen from: halogen, carboxyl, sulfonate,ammonium, alcohol, ether, alkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxy, haloalkyl, arylalkyl, heteroarylalkyl andarylheterocycloalkyl.

By way of example of an alkyl radical, mention may be made of one ofthose chosen from the group comprising ethyl, 2-hydroxyethyl,3-hydroxypropyl, NaSO₃CH₂CH₂—, HOOC—CH₂CH₂—, HOOC—CH₂— orCH₃CH₂COOCH₂CH₂—.

By way of example of an aryl or arylalkyl radical, mention may be madeof one of those chosen from the group comprising benzyl (C₆H₅—CH₂—),4-methoxybenzyl (p-MeO-C₆H₅—CH₂—), 2,4,6-trimethoxybenzyl,4-methylbenzyl, phenyl (C₆H₅—), p-(HOOC—CH₂)—C₆H₄—, p-CF₃—C₆H₄—,p-F—C₆H₄—, p-C₅-C₆H₄—, p-Br—C₆H₄—, p-I—C₆H₄—, p-NO₂—C₆H₄—, p-Me-C₆H₄—and p-HO—CH₂—C₆H₄—.

The reaction between the peptide C^(α)-amide of formula (II) and thethiol R′—SH may be carried out in the presence of an excess of thiol intrifluoroacetic acid (TFA), acetic acid or formic acid or in an aqueousbuffer, optionally with an organic cosolvent, urea or guanidiniumchloride added thereto, so as to ensure the solubilization of thepeptide C^(α)-amide of formula (II) or of the thiol R′—SH.

The pH of the aqueous buffer may be from 0 to 8 and preferably from 1 to4.

By way of example of an organic cosolvent, mention may be made of one ofthose chosen from the group comprising acetonitrile (MeCN), methanol(MeOH), isopropanol (iPrOH), dimethylformamide (DMF) andN-methyl-2-pyrrolidone (NMP).

Preferably, the reaction may be carried out in a 7:3 waterMeCN mixture,in the presence of 0.1% vv TFA and of 50 equivalents of a thiol R′SH.

In one particular mode of preparation of a peptide C^(α)-thioester offormula (IX), D represents a solid support suitable for Fmoc SPPS and Cis absent or represents an arm that can be used for Fmoc SPPS andpreferably an arm that is stable with respect to TFA, such as the PAM orMBHA arm. In this particular mode, the peptide C^(α)-thioester offormula (IX) is obtained by means, firstly, of a treatment of thepeptidyl resin with TFA in order to cleave the protective groups of theside chains of Peptide1 that were used during the step in which Peptide1was elongated by Fmoc SPPS, without detaching it from the solid support.Then, secondly, the thioesterification of the supported deprotectedpeptide obtained after the treatment with TFA is carried out by means ofthe thiol R′—SH. This also has the effect of detaching the peptideC^(α)-thioester of formula (IX) from the solid support.

Advantageously according to the invention, the peptide C^(α)-amide asdefined above may be used to prepare a peptide C^(α)-thioester. Asubject of the invention is therefore also the use of a peptideC^(α)-amide as defined above, for preparing a peptide C^(α)-thioester.

The invention also relates to a process for preparing a peptide ofgeneral formula (X):

Peptide1-Yaa-Peptide2-R₁₅  (X)

-   -   in which:    -   Peptide1 is as previously defined,    -   Yaa is an amino acid residue originating from an amino acid of        formula H-Yaa-OH chosen from the group comprising a cysteine, a        homocysteine, a β-mercaptovaline, a β-mercaptoleucine, a        β-mercaptoisoleucine, a β-mercaptophenylalanine, a        β-mercaptolysine, a β-mercaptoproline, a γ-mercaptovaline, a        γ-mercaptoisoleucine, a γ-mercaptoleucine, a γ-mercaptolysine, a        γ-mercaptoproline, or an amino acid substituted on its nitrogen        atom N^(a) with a group containing a β- or γ-aminothiol        function, said group being chosen from the group comprising:

-   -   Peptide2=(Xaa)₁, with 1=an integer ranging from 1 to 60,        preferably from 4 to 40,    -   Xaa represents, independently of one another, an amino acid        residue originating from an amino acid of formula H-Xaa-OH, and        when 1 is greater than or equal to 2, each of said Xaa is        connected to its neighbor Xaa via a peptide bond,    -   R₁₅ represents —OH, —NH₂ or a radical of general formula (I) as        defined above, in which X═S and R₁ is a protective group for        sulfur which is stable with respect to treatment with TFA and        stable under NCL conditions,    -   characterized in that:    -   a peptide C^(α)-amide of general formula (II) as defined above,        in which R1=H or a group which is labile under NCL native        chemical ligation conditions, is reacted, by means of an NCL        reaction,    -   with a peptide possessing an N-terminal β- or γ-aminothiol        residue of general formula (XI):

H-Yaa-Peptide2-R₁₅  (XI)

-   -   in which Yaa, R₁₅ and Peptide2 are as defined above,    -   in order to obtain        -   the peptide of general formula (X) as defined above and        -   the compound of formula (Ia) as defined above.

Advantageously according to the invention, the peptide C^(α)-amides havecrypto-thioester properties and, in this regard, can therefore be useddirectly in an NCL native chemical ligation reaction, without beingconverted beforehand to peptide C^(α)-thioesters. A subject of theinvention is therefore also the use of a peptide C^(α)-amide as definedabove, in an NCL native chemical ligation reaction for preparing apeptide or a protein, in particular of therapeutic interest.

A subject of the present invention is also a process for preparing apeptide of general formula (XII):

Peptide1-Yaa₂-Peptide2- . . .-Yaa_(q-1)-Peptide(q−1)-Yaa_(q)-Peptide(q)-R₁₅  (XII)

-   -   in which:    -   R₁₅ is as previously defined,    -   q is an integer ranging from 3 to 10,    -   each Yaa (Yaa₂, . . . , Yaa_(q-1) and Yaa_(q)) represents,        independently of the others, an amino acid residue as previously        defined,    -   each peptide of formula (XIII) above (Peptide2 . . .        Peptide(q−1) and Peptide(q)), except Peptide1, represents,        independently of the others, (Xaa)₁ as previously defined with        respect to Peptide2,    -   characterized in that:    -   a peptide of formula (II) as previously defined, in which R₁═H        or a group which is labile under NCL native chemical ligation        conditions, is reacted, by means of a first NCL reaction,    -   with a peptide of general formula (XIa):

H-Yaa₂-Peptide2-R₁₆  (XIa)

-   -   in which:    -   Yaa₂ is equal to Yaa as previously defined, and Peptide2 is as        previously defined, R₁₆ represents a radical of formula (I) in        which R₁ represents a protective group which is stable under NCL        conditions,    -   in order to obtain a peptide of general formula (XIII)

Peptide1-Yaa₂-Peptide2-R₁₆  (XIII)

-   -   in which:    -   Yaa₂, Peptide1, Peptide2 and R₁₆ are as previously defined,    -   and a compound of formula (Ib) as previously defined.

The peptide of general formula (XIII) which exhibits the R₁₆ radical aspreviously defined is then subjected to a deprotection under theconditions known to those skilled in the art in order to obtain thepeptide of general formula (XIIIa)

Peptide1-Yaa₂-Peptide2-R₁₇  (XIIIa)

-   -   in which:    -   R₁₇ represents a radical of formula (I) in which R₁ represents a        hydrogen.

The compound (XIIIa) thus obtained is:

-   -   either converted beforehand to a peptide thioester and then        reacted with a peptide of general formula (Xlb):

H-Yaa₃-Peptide3-R₁₆  (XIb),

-   -   or directly reacted with the peptide of formula (XIb), by        exploiting the crypto-thioester properties of the compound        (XIIIa)

This successive assembly is iteratively continued, by repeating (q−3)times the successive NCL and deprotection steps as described above.

The final compound of general formula (XII) is obtained by means of afinal assembly making it possible to add the Peptide(q).

This final assembly is carried out:

-   -   either in two steps, namely prior conversion to thioester and        then reaction with a peptide of general formula (XIc):

H-Yaa_(q)-Peptide(q)-R₁₅  (XIc),

-   -   or by direct reaction with said peptide of formula (XIc).

When q is an integer equal to 3, then the peptide of formula (XII) isrepresented by the following formula (XII):

Peptide1-Yaa₂-Peptide2-Yaa₃-Peptide3-R₁₅  (XII).

The compound of formula (XIa) is a peptide C^(α)-amide which does nothave crypto-thioester properties owing to the masking of its thiolfunction by an R₁ radical representing a protective group for sulfurwhich is stable with respect to treatment with TFA and stable under NCLconditions.

A preferred R₁ group of the radical (I) present in said compound (XIa)is more particularly a group chosen from the group comprisingacetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), benzamidomethyl(B am), allyloxycarbonylaminomethyl (Allocam), phthalimidomethyl (Pim),2-(trimethylsilyl)ethyl, t-butyl (tBu), 2,4,6-trimethylbenzyl,quinolinylmethyl (Qm), diphenyl-4-pyridylmethyl, 1-adamantyl,benzyloxymethyl (BOM), 2-tetrahydropyranyl (Thp) and benzylthiomethyl.

According to one preferred embodiment of the process for preparing thepeptide of general formula (XII), Peptide1 is grafted beforehand onto ahydrocompatible solid support, for instance a support chosen from thegroup comprising Pega®, Chemmatrix™, agarose, sepharose and controlledpore glass (CPG) microparticles. More particularly, Peptide1 is graftedonto a solid support via a linker which can be cleaved after the (q−1)steps of NCL native chemical ligation, said linker preferably being anN-terminal arm as described in document WO 2011058188. Other ligationreactions (optionally requiring other types of precursors) may also becombined with NCL in the same strategy of multiple ligations on a solidsupport from N toward C.

The invention will be understood more clearly in the light of thenonlimiting and purely illustrative examples which follow. FIGS. 6 to 26make it possible to illustrate said examples below.

FIGS. 1 to 5, relating to the prior art, have been commented upon in theintroduction of the present application.

FIG. 1 represents the steps of solid phase peptide synthesis (SPPS).

FIG. 2 represents the protective groups (Boc and Fmoc) for amines usedin SPPS.

FIG. 3 represents an “NCL” native chemical ligation reaction between apeptide C^(α)-thioster (compound (a)) and a peptide bearing anN-terminal cysteine (compound (b)).

FIG. 4 represents the use of peptide C^(α)-N-alkyl(oraryl)-N-(β-mercaptoalkyl)-amides (e) for:

-   -   the synthesis of peptide C^(α)-thioesters (a) (by acyl transfer        from N to S) (FIGS. 4-1 and 4-2),    -   direct use in NCL (FIG. 4-3).

FIG. 5-1 represents a scheme of the principle of the synthesis ofpeptide C^(α)-β-mercapto-amides of type (e), detailing the difficultstep of N-acylation of the secondary amine.

FIG. 5-2 is a comparison between the acylation of a secondary aminebearing a 2-hydroxybenzyl group (j) and that of a secondary aminebearing a 2-methoxybenzyl group (m), and illustrates more particularlythe principle of the assistance, by a 2-hydroxybenzyl group, of theacylation of a secondary amine.

FIG. 6 is a synthesis scheme illustrating the preparation of sixcompounds of the invention corresponding to general formula (Ia)(compounds 3a, 3b, 3c, 3′a, 3′b and 3′c).

FIG. 7 is a synthesis scheme illustrating the preparation of a peptideC^(α)-amide of general formula (II) (compounds 5 and 5′) from a compoundof general formula (Ia) (compound 3a).

FIG. 8 represents the chromatogram of an HPLC/MS analysis of the peptide2′ represented in FIG. 6.

FIGS. 9, 10 and 11 represent the chromatograms of respective HPLC/MSanalyses of the peptides 3′a, 3′b and 3′c represented in FIG. 6.

FIGS. 12 and 13 represent the chromatograms of respective HPLC/MSanalyses of the peptides 4′a and 4′b represented in FIG. 7.

FIG. 14 represents the chromatogram of an HPLC/MS analysis of thepeptide 5′ represented in FIG. 7.

FIG. 15 represents the use of a peptide C^(α)-amide of general formula(II) (compound 5′) for obtaining a peptide C^(α)-thioester (compound 6).

FIG. 16 represents the use of a peptide C^(α)-amide of general formula(II) (compound 5′) in an NCL reaction for obtaining a model peptide(compound 9) (reactivity of crypto-thioester type).

FIG. 17 is a synthesis scheme illustrating the preparation of threecompounds of the invention corresponding to general formula (Ia)(compounds 12, 13 and 13′).

FIG. 18 represents the chromatogram of an HPLC/MS analysis of compound13′ represented in FIG. 17.

FIG. 19 is a synthesis scheme illustrating the preparation of a peptideC^(er)-amide of general formula (II) (compounds 14 and 14′) from acompound of general formula (Ia) (compound 12).

FIG. 20 represents the chromatogram of an HPLC/MS analysis of thepeptides 14′ and 14″ represented in FIG. 19.

FIG. 21 is a synthesis scheme illustrating the preparation of a peptideC^(er)-amide of general formula (II) (compounds 15 and 15′) from acompound of general formula (Ia) (compound 13).

FIG. 22 represents the chromatogram of an HPLC/MS analysis of thepeptide 15′ represented in FIG. 21.

FIG. 23 represents the use of the peptides 14′/14″ in an NCL nativechemical ligation reaction so as to give a peptide 17, followed by aone-pot oxidative folding in order to obtain a peptide 19.

FIG. 24 represents the chromatogram of an HPLC/MS analysis of thepeptide 16 represented in FIG. 23.

FIG. 25 represents the chromatogram of an HPLC/MS analysis of thepeptide 17 represented in FIG. 23.

FIG. 26 represents the chromatogram of an HPLC/MS analysis of thepeptide 19 represented in FIG. 23.

EXAMPLES

In examples 1 and 2 described below, in the radical of formula (I) or inthe compound (Ia):

-   -   X represents a sulfur atom,    -   R₁ represents a hydrogen atom or a trityl (Trt) group,    -   R₂ represents a group —B—C-D in which:        -   B represents —CO-Leu-Tyr-Arg-Ala-Gly-O—, C is absent and D            represents a hydrogen atom, or        -   B represents —CO-Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly-O—, C            represents a Wang arm (—CH₂—C₆H₄—O—CH₂—) and D represents a            solid support such as a resin of polystyrene type,    -   R₃, R₄ and R₅ each represent a hydrogen atom,    -   m is equal to n which is equal to 0,    -   A representing an aryl group of general formula:

-   -   in which X₁ represents C—OH, each of X₂, X₃ and X₅ represents CH        and X₄ represents CH, C—OCH₃ or C—NO₂,    -   and, if it is the compound (Ia), R₂₀ represents a hydrogen atom.

When D represents a hydrogen atom, this means that the radical (I) isnot attached to a solid support. In this case, R₁ represents H and R₂represents a group —B—C-D in which B represents—CO-Leu-Tyr-Arg-Ala-Gly-O—, C is absent and D represents a hydrogenatom.

When D represents a solid support, this means that the radical (I) isattached to said solid support (said solid support being an integralpart of the definition of the radical (I)). In this case, R₁ representstrityl (Trt) and R₂ represents a group —B—C-D in which B represents—CO-Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly-O—, C represents a Wang arm(—CH₂—C₆H₄—O—CH₂—) and D represents a solid support such as a resin ofpolystyrene type.

The products obtained were analyzed by HPLC on a column: nucleosil C18300 Å 5 μm, 4.6×250 mm, gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min, flow rate: 1 mlmin, followed by UV detection (λ=276 nm or 360nm) and then MALDI-TOF mass spectrometry.

Example 1 Preparation of a Peptide C^(α)-Amide of General Formula (II)

1) Synthesis of a Compound of General Formula (Ia)

The synthesis of a compound of general formula (Ia) is illustrated inFIG. 6. Compounds 3a, 3b, 3c and 3′a, 3′b and 3′c correspond to generalformula (Ia) in which:

-   -   for compounds 3a, 3b and 3c, X represents S, R₁ represents Trt,        R₃, R₄ and R₅ represent H, m is equal to n which is equal to 0,        R₂₀ represents a hydrogen atom, R₂ represents

and A represents respectively:

-   -   for compounds 3′a, 3′b and 3′c, X represents S, R₁ represents H,        R₃, R₄ and R₅ represent H, m is equal to n which is equal to 0,        R₂₀ represents a hydrogen atom, R₂ represents        —CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents respectively:

Synthesis of the Peptidyl Resin 2

The synthesis scheme is illustrated in FIG. 6.

Compound 1 corresponds to the formula H-E-O—C-D′ as defined above inwhich:

-   -   E represents Leu-Tyr(t-Bu)-Arg(Pbf)-Ala-Gly,    -   C represents a Wang arm and D′ represents a resin of polystyrene        type.

Compound 2 corresponds to general formula (IVa) as defined above:

-   -   in which    -   X represents S, R₁ represents Trt, R″₃, R″₄, R″₅, R″₆ and R″₇        represent H, m is equal to 0, and R″₂ represents:

The peptidyl resin 2 of formula:

-   -   was synthesized under standard conditions (ABI 433 synthesizer,        Fastmoc program). An aliquot of resin is treated for 2 h with        87.5:5:5:2.5 CF₃COOHphenolH₂Otri-isopropylsilane so as to detach        and deprotect the peptide 2 in order to obtain the peptide 2′,        which is precipitated by dilution in a 1:1 petroleum ether/Et₂O        mixture, washed (Et₂O), dried, and then analyzed by HPLC and        mass spectrometry (see FIG. 8).

Peptide 2′ (see FIG. 8):

Empirical formula: C₂₉H₄₇N₉O₈S;

HPLC: t_(R)=14.41 min (C18, gradient: 5-50% MeCNH₂O+0.1% TFA over thecourse of 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z observed=682.0 ([MH]⁺ calculated forC₂₉H₄₇N₉O₈S=682.3).

Reductive Aminations

The synthesis scheme is illustrated in FIG. 6.

0.1 mmol of the peptidyl resin 2 of formula:

-   -   is solvated in the mixture dimethylformamide/methanolacetic acid        (9:9:2).

10 equivalents of aldehyde (of general formula VII) as represented inFIG. 6 are then dissolved in 4 ml of dimethylformamide/methanol (1:1)and are added to the peptidyl resin 2. The resulting suspension isstirred for 45 minutes. The resin is then washed with thedimethylformamide/methanol mixture (1:1).

20 equivalents of NaBH₃CN are dissolved in 4 ml ofdimethylformamide/methanolacetic acid (9:9:2) and then added to theresin. The resulting suspension is stirred for 30 minutes. The resin iswashed with the dimethylformamide/methanolacetic acid mixture (9:9:2).An aliquot of resin is treated for 2 h with 87.5:5:5:2.5TFA/PhOH/H₂O/i-Pr₃SiH so as to detach and deprotect the peptide 3a, 3bor 3c in order to obtain respectively the peptide 3′a, 3′b or 3′c, whichis precipitated by dilution in a 1:1 petroleum ether/Et₂O mixture,washed (Et₂O), dried and then analyzed by HPLC and mass spectrometry(see FIGS. 9, 10 and 11).

Peptide 3′a (FIG. 9):

Empirical formula: C₃₆H₅₂N₁₀O₁₁S;

HPLC: t_(R)=20.44 min (C18, gradient: 5-50% MeCNH₂O+0.1% TFA over thecourse of 30 min);

UV detection (λ=360 nm);

MS (MALDI-TOF): m/z observed=833.3 ([M1-1]⁺ calculated forC₃₆H₅₂N₁₀O₁₁S=833.4).

Peptide 3′b (FIG. 10):

Empirical formula: C₃₆H₅₃N₉O₉S;

HPLC: t_(R)=19.17 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=788.3 ([MH]⁺ calculated forC₃₆H₅₃N₉O₉S=788.4).

Peptide 3′c (FIG. 11):

Empirical formula: C₃₇H₅₅N₉O₁₀S;

HPLC: t_(R)=19.59 min (C18, gradient: 5-50% MeCNH₂O+0.1% TFA over thecourse of 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=818.4 ([MH]⁺ calculated forC₃₇H₅₅N₉O₁₀5=818.4).

2) Synthesis of a Peptide C^(α)-Amide of General Formula (II)

The synthesis of a peptide C^(α)-amide of general formula (II) isillustrated in FIG. 7. Compounds 5 and 5′ correspond to general formula(II) in which:

-   -   for compound 5, X represents S, R₁ represents Trt, R₃, R₄ and R₅        represent H, Peptide1 represents Ac-Tyr(tBu)-Arg(Pbf)-Phe-Gly, m        is equal to n which is equal to 0, R₂ represents

and A represents

-   -   for compound 5′, X represents S, R₁ represents H, R₃, R₄ and R₅        represent H, Peptide1 represents Ac-Tyr-Arg-Phe-Gly-, m is equal        to n which is equal to 0, R₂ represents        —CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents:

Coupling of Fmoc-Glycine onto the Supported N-Alkyl Cysteine 3a

The coupling of Fmoc-glycine onto the peptidyl resin 3a is illustratedin FIG. 7.

The coupling of Fmoc-glycine onto the peptidyl resin 3a is carried outunder standard conditions (ABI 433, Fastmoc program). The peptidyl resin3a is solvated in DMF. 10 equivalents of protected amino acid(Fmoc-Gly-OH as represented in FIG. 7) are then activated with 10equivalents of HBTU O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) in the presence of 10 equivalents of HOBt(1-hydroxybenzotriazole) and 20 equivalents of DIEA(diisopropylethylamine) and then added to the resin, which is thenstirred for 30 minutes at ambient temperature. The resin is rinsed withDMF and CH₂Cl₂ and then the Fmoc group is cleaved using piperidine (20%in NMP), three successive treatments of five minutes each. The resin isrinsed with DMF and CH₂Cl₂. An aliquot of resin 4a thus obtained istreated for 2 h with 87.5:5:5:2.5 TFA/PhOH/H₂O/i-Pr₃SiH so as to detachand deprotect the peptide 4a and to obtain the peptide 4′a, which isanalyzed by HPLC and mass spectrometry (FIG. 12).

Peptide 4′a (FIG. 12):

Molecular formula: C₃₈H₅₅N₁₁O₁₂S;

HPLC: t_(R)=20.53 min (C18, gradient: 5-50% MeCNH₂O+0.1% TFA over thecourse of 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=890.4 ([MH]⁺ calculated forC₃₈H₅₅N₁₁O₁₂S=890.4).

Coupling of Fmoc-Alanine onto the Supported N-Alkyl Cysteine 3a

The coupling of Fmoc-alanine onto the peptidyl resin 3a is illustratedin FIG. 7.

The peptidyl resin 3a is solvated in DMF. 10 equivalents of protectedFmoc-alanine (Fmoc-Ala-OH as represented in FIG. 7) are then activatedwith 10 equivalents of HBTU in the presence of 10 equivalents of HOBtand 20 equivalents of DIEA in DMF and of the peptidyl resin 3a for 30minutes at ambient temperature. This step is carried out twice. Theresin is rinsed with DMF and DCM and then the amine is deprotected withpiperidine (20% in NMP), three times for five minutes. The resin isrinsed with DMF and DCM. An aliquot of resin 4b thus obtained is treatedfor 2 h with 87.5:5:5:2.5 TFA/PhOH/H₂O/i-Pr₃SiH so as to detach anddeprotect the peptide 4b and to obtain the peptide 4′b, which isanalyzed by HPLC and mass spectrometry (FIG. 13).

Peptide 4′b (FIG. 13):

Molecular formula: C₃₉H₅₇N₁₁O₁₂S;

HPLC: t_(R)=21.50 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min DAD detection);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=904.5 ([MFI]⁺ calculated forC₃₉H₅₇N₁₁O₁₂S=904.4).

Elongation of the Peptide 5′ Using the Compound 4a Obtained

The elongation of the peptide 5′ is represented in FIG. 7.

The continuation of the elongation of the peptide is carried out understandard conditions (ABI 433, Fastmoc program) with the peptidyl resin4a. An aliquot of resin 5 is treated for 2 h with 87.5:5:5:2.5TFA/PhOH/H₂O/i-Pr₃SiH so as to obtain the peptide 5′ which isprecipitated by dilution in a 1:1 petroleum ether/Et₂O mixture, washed(Et₂O), dried and then analyzed by HPLC and mass spectrometry (FIG. 14).

Peptide 5′ (FIG. 14):

Molecular formula: C₃₉H₅₇N₁₁O₁₂S;

HPLC: t_(R)=21.50 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min DAD detection);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=904.5 ([MH]⁺ calculated forC₃₉H₅₇N₁₁O₁₂S=904.4).

Example 2 Use of the Peptide C^(α)-Amide of General Formula (II)

1) in a Thioesterification Reaction in an Acidic Medium

The use of a peptide C^(α)-amide 5′ of general formula (II) in athioesterification reaction is illustrated in FIG. 15.

2.5 μmol of peptide 5′ (peptide of general formula (II)) and 100 μmol ofmercaptophenylacetic acid (namely a thiol of general formula R′—SH withR′ representing pHOOC—CH₂—C₆H₄— as represented in FIG. 15) are dissolvedin 1 ml of the mixture H₂OMeCN (7:3)+0.1% TFA. The reaction mixture isstirred for 48 hours at ambient temperature. The progression of thereaction is monitored by HPLC (FIG. 15).

The desired peptide C^(α)-thioester of formula 6 and compound 7corresponding to general formula (Ia) in which:

X represents S, R₁ represents H, R₃, R₄ and R₅ represent H, R₂₀represents H, m is equal to n which is equal to 0, R₂ represents—CO-Leu-Tyr-Arg-Ala-Gly-OH, and A represents:

are thus obtained.

This compound of formula (Ia) is particularly advantageous owing to itscharacteristic absorption in the ultraviolet range (λmax˜400-450 nm):HPLC analysis with detection in this wavelength range makes it possibleto easily detect it and characterize it, which will also allow thoseskilled in the art to conclude that the peptide C^(α)-thioester offormula (IX) as defined above has indeed been formed.

Peptide C^(α)-thioester 6 (FIG. 15):

2) in an NCL Native Chemical Ligation Reaction

The use of a peptide C^(α)-amide 5′ of general formula (II) in a nativechemical ligation reaction is illustrated in FIG. 16.

The peptide C^(α)-amide of general formula (II) (compound 5′), by virtueof its crypto-thioester properties, can be used directly in an NCLnative chemical ligation reaction, without converting it beforehand tothe peptide C^(α)-thioester (FIG. 16).

2.5 μmol of crude crypto-thioester peptide 5′ and 1.5 equivalents ofcysteinyl peptide 8 are dissolved in 1 ml of degassed solutioncontaining 50 mM of mercaptophenylacetic acid, 20 mM oftriscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7. Thereaction mixture is stirred for 24 h at 37° C. under argon.

A peptide 9 and a compound 7 of general formula (Ia) are thus obtained.

Peptide 9 (FIG. 16): Ac-Tyr-Arg-Phe-Gly-Cys-Leu-Tyr-Arg-Ala-Gly-OH

In examples 3 and 4 described below, in the radical of formula (I) or inthe compound (Ia):

-   -   X represents a sulfur atom,    -   R₁ represents a hydrogen atom, a trityl (Trt) group, or a        tert-butylsulfanyl (StBu) group,    -   R₂ represents a group —B—C-D in which:        -   B represents —CO—NH—, where        -   C is absent and D represents a hydrogen atom, or        -   C represents a Rink arm            (—CH[2,4-di-MeO-C₆H₄]-C₆H₄—O—CH₂—CO—NH—CH₂) and D represents            a solid support such as a resin of ChemMatrix or polystyrene            type,    -   R₃, R₄ and R₅ each represent a hydrogen atom,    -   m is equal to n which is equal to 0,    -   A representing an aryl group of general formula:

-   -   -   in which X₁ represents C—OH, each of X₂, X₃ and X₅            represents CH and X₄ represents C—NO₂,

    -   and, if it is the compound (Ia), R₂₀ represents a hydrogen atom.

When D represents a hydrogen atom, this means that the radical (I) isnot attached to a solid support. In this case, R₁ represents H or atert-butylsulfanyl (StBu) group and R₂ represents a group —B—C-D inwhich B represents —CO—NH—, C is absent and D represents a hydrogenatom.

When D represents a solid support, this means that the radical (I) isattached to said solid support (said solid support being an integralpart of the definition of the radical (I)). In this case, R₁ representsa trityl (Trt) group or a tert-butylsulfanyl (StBu) group and R₂represents a group —B—C-D in which B represents —CO—NH—, C represents aRink arm (—CH[2,4-di-MeO-C₆H₄]-C₆H₄—O—CH₂—CO—NH—) and D represents asolid support such as a resin of ChemMatrix or polystyrene type.

The products obtained were analyzed by HPLC on a column: nucleosil C18300 λ 5 μm, 4.6×250 mm, gradient: 5-50% MeCN/H₂O+0.1% TFA over thecourse of 30 min, flow rate: 1 ml/min, followed by UV detection (λ=276nm or 320 nm) and then by MALDI-TOF mass spectrometry.

Example 3 Preparation of Peptide C^(α)-Amide of General Formula (II)

1) Synthesis of a Compound of General Formula (Ia)

The synthesis of a compound of general formula (Ia) is illustrated inFIG. 17.

Compounds 12, 13 and 13′ correspond to general formula (Ia) in which:

-   -   for compounds 12 and 13, X represents S, R₃, R₄ and R₅ represent        H, m is equal to n which is equal to 0, R₂₀ represents a        hydrogen atom,    -   R₂ represents

A represents:

-   -   and R₁ represents Trt (12), or StBu (13),    -   for compound 13′, X represents S, R₁ represents StBu, R₃, R₄ and        R₅ represent H, m is equal to n which is equal to 0, R₂₀        represents a hydrogen atom, R₂ represents —CO—NH₂, A represents:

Synthesis of the Cysteinyl Resins 10 and 11

The synthesis scheme is illustrated in FIG. 17.

Compounds 10 and 11 correspond to general formula (IVa) as definedabove:

-   -   in which    -   X represents S, R″₃, R″₄, R″₅, R″₆ and R″₇ represent H, m is        equal to 0, R″₂        represents

and R₁ represents Trt (10) or StBu (11).

The cysteinyl resins 10 and 11 of respective formula:

-   -   were synthesized under standard conditions (ABI 433 synthesizer,        Fastmoc program).

Reductive Aminations

The synthesis scheme is illustrated in FIG. 17.

0.1 mmol of the peptidyl resin 10 or 11 as defined respectively above issolvated in the mixture dimethylformamide/methanol/acetic acid (9:9:2).

167 mg (10 equivalents) of 2-hydroxy-5-nitrobenzaldehyde dissolved in 4ml of dimethylformamide/methanol (1:1) are added to the resin. Theresulting suspension is stirred for 45 minutes. The resin is then washedwith the dimethylformamide/methanol (1:1) mixture.

126 mg (20 equivalents) of NaBH₃CN dissolved beforehand in 4 ml ofdimethylformamide/methanolacetic acid (9:9:2) are added to the resin.The resulting suspension is stirred for 30 minutes. The resin is washedwith the dimethylformamide/methanolacetic acid (9:9:2) mixture. Analiquot of the resin 13 is treated for 2 h with 92.5:5:2.5TFAH₂O/i-Pr₃SiH in order to obtain the compound 13′, which is analyzedby HPLC and mass spectrometry after evaporation of the TFA (see FIG.18).

Compound 13′ (FIG. 18):

Empirical formula: C₁₄H₂₁N₃O₄S₂;

HPLC: t_(R)=23.4 min (C18, gradient: 5-50% MeCNH₂O+0.1% TFA over thecourse of 30 min);

UV detection (λ=320 nm);

MS (ESI+): m/z observed=360.1 ([MH]⁺ calculated for C₁₄H₂₂N₃O₄S₂=360.1).

2) Synthesis of a Peptide C^(α)-Amide of General Formula (II)

The synthesis of a peptide C^(α)-amide of general formula (II) isillustrated in FIG. 19. Compounds 14, 14′, 15 and 15′ correspond togeneral formula (II) in which:

-   -   for compound 14, X represents S, R₁ represents Trt, R₃, R₄ and        R₅ represent H, Peptide1 represents        H-Ala-Ser(tBu)-Cys(Trt)-Asn(Trt)-Gly-Val-Cys(Trt)-Pro-Phe-Glu(OtBu)-Met-Pro-Pro-Cys(Trt)-Gly-Thr(tBu)-Ser(tBu)-Ala-,        m is equal to n which is equal to 0, R₂ represents

and A represents:

-   -   for compound 14′, X represents S, R₁ represents H, R₃, R₄ and R₅        represent H, Peptide1 represents        H-Ala-Ser-Cys-Asn-Gly-Val-Cys-Pro-Phe-Glu-Met-Pro-Pro-Cys-Gly-Thr-Ser-Ala-,        m is equal to n which is equal to 0,    -   R₂ represents —CO—NH₂, and A represents:

-   -   for compound 15, X represents S, R₁ represents StBu, R₃, R₄ and        R₅ represent H, Peptide1 represents        H-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Gly-, m is equal to n which is equal        to 0, R₂ represents

and A represents

-   -   for compound 15′, X represents S, R₁ represents StBu, R₃, R₄ and        R₅ represent H, Peptide1 represents        H-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Gly-, m is equal to n which is equal        to 0, R₂ represents —CO—NH₂, and A represents:

Elongation of the Peptide 14′ Using Compound 12

The elongation of the peptide 14′ is represented in FIG. 19.

The elongation of the peptide is carried out under standard conditions(ABI 433, Fastmoc program, HCTU as coupling agent) with the resin 12, bycarrying out twice in a row the coupling of the first amino acidFmoc-Ala-OH before deprotecting the Fmoc group and continuing theelongation.

The peptidyl resin 14 obtained is treated for 2 h with 87.5:5:5:2.5TFA/PhOH/H₂O/i-Pr₃SiH so as to obtain the peptide 14′ which isprecipitated by dilution in a 1:1 petroleum ether/Et₂O mixture, washed(Et₂O) and then dried under reduced pressure. The precipitate is takenup in demineralized water and lyophilized. It is then analyzed by HPLCand mass spectrometry (FIG. 21). The peptide amide 14′ is obtained bymixing with the peptide thioester 14″ (the two compounds are inequilibrium in solution).

Peptide 14′ (FIG. 20):

Molecular formula: C₈₅H₁₂₆N₂₃O₃₁S₅;

HPLC: (gradient: 5-50% MeCNH₂O+0.1% TFA over the course of 30 min UVdetection, λ=276 nm) t_(R)=25.3 min;

MS (ESI+): m/z obtained=1055.9 ([M+2H]²⁺).

Peptide 14″ (FIG. 20):

Molecular formula: C₈₅H₁₂₆N₂₃O₃₁S₅;

HPLC: t_(R)=23.8 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min;

UV detection (λ=276 nm);

MS (ESI+): m/z obtained=1055.9 ([M+2H]²⁺).

Elongation of the Peptide 15′ Using Compound 13

The elongation of the peptide 15′ is represented in FIG. 21.

The elongation of the peptide is carried out under standard conditions(ABI 433, Fastmoc program, HCTU as coupling agent) with the resin 13, bycarrying out twice in a row the coupling of the first amino acidFmoc-Ala-OH before deprotecting the Fmoc group and continuing theelongation.

The peptidyl resin 15 obtained is treated for 2 h with 97.5:5:5:2.5TFA/i-Pr₃SiH so as to obtain the peptide 15′ which is precipitated bydilution in a 1:1 petroleum ether/Et₂O mixture, washed (Et₂O), dried andthen analyzed by HPLC and mass spectrometry (FIG. 21).

Peptide 15′ (FIG. 22):

Molecular formula: C₄₃H₆₅N₁₁O₁₁S₂;

HPLC: t_(R)=31.0 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min);

UV detection (λ=320 nm);

MS (MALDI-TOF): m/z obtained=976.4 ([MH]⁺ calculated forC₄₃H₆₅N₁₁O₁₁S₂=976.4).

Example 4 Use of the Peptide C^(α)-Amide of General Formula (II) in aNative Chemical Ligation (NCL) Reaction

The use of a peptide C^(α)-amide 14′ of general formula (II) in a nativechemical ligation reaction is illustrated in FIG. 23.

The peptide C^(α)-amide of general formula (II) (compound 14′), byvirtue of its crypto-thioester properties, can be used directly in anNCL native chemical ligation reaction, without converting it beforehandto the peptide C^(α)-thioester (FIG. 23).

Preparation of the Cysteinyl Peptide 16 Partner

The peptide 16 (H—CRCIPVGLIGYCRNPSG-OH) was synthesized under standardconditions (ABI 433 synthesizer, Fastmoc program, coupling agent: HCTU).The resin obtained after automated elongation is treated for 2 h with87.5:5:5:2.5 TFA/PhOH/H₂O/i-Pr₃SiH so as to detach and deprotect thepeptide 16, which is precipitated by dilution in a 1:1 petroleumether/Et₂O mixture, washed (Et₂O) and then dried. The precipitate istaken up in demineralized water and then lyophilized. It is thenanalyzed by HPLC and mass spectrometry (FIG. 24).

Peptide 16 (FIG. 24):

Molecular formula: C₈₁H₁₃₅N₂₅O₂₂S₃;

HPLC: t_(R)=24.3 min (gradient: 5-50% MeCNH₂O+0.1% TFA over the courseof 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=1906.9 ([MH]+ calculated forC₈₁H₁₃₆N₂₅O₂₂S₃=1906.9).

Native Chemical Ligation Between the Peptides 14′/14″ and 16

2.5 μmol of crude crypto-thioester peptide 14′/14″ and 1.5 equivalentsof crude cysteinyl peptide 16 are dissolved in 1 ml of a deoxygenatedsolution containing 25 mM of mercaptophenylacetic acid, 50 mM oftriscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7. Thereaction mixture is stirred for 3 h at 37° C. under an argon atmosphere.A peptide 17 and a compound 18 of general formula (Ia) are thusobtained. The peptide 17 is purified by semi-preparative HPLC and thenlyophilized. It is obtained with a yield of 65%.

Peptide 17 (FIG. 25):

Molecular formula: C₁₅₆H₂₄₉N₄₅O₄₈S₇;

HPLC: t_(R)=14.6 min (gradient: 30-45% MeCNH₂O+0.1% TFA over the courseof 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=3745.6 ([M+H]⁺ calculated forC₁₅₆H₂₅₀N₄₅O₄₈S₇=3745.6).

Native Chemical Ligation Between the Peptides 14′/14″ and 16 Followed bya One-Pot Oxidative Folding

2.5 μmol of crude crypto-thioester peptide 14′/14″ and 3.75 μmol (1.5equivalents) of crude cysteinyl peptide 16 are dissolved in 1 ml of adeoxygenated solution containing 25 mM of mercaptophenylacetic acid, 50mM of triscarboxyethylphosphine and 200 mM of phosphate buffer, pH=7.The reaction mixture is stirred for 3 h at 37° C. under an argonatmosphere. The reaction mixture is directly used in a step of oxidativefolding of the ligation product 17 (final concentration of 17: 13 μM) bydilution in a 1:1 mixture of isopropanol and of a buffer containing 0.2mM of Tris, pH 8.6, 2 mM of EDTA, 1.82 mM of glutathione (GSH) and 0.78mM of oxidized glutathione (GSSG), and stirring of the resultingsolution for 24 hours at 4° C. The mini-protein 19 comprising threedisulfide bridges is purified by semi-preparative HPLC and thenlyophilized. 19 is obtained with a yield of 55%.

Peptide 19 (FIG. 26):

Molecular formula: C₁₅₆H₂₄₃N₄₅O₄₈S₇;

HPLC: t_(R)=28.4 min (gradient: 30-45% MeCNH₂O+0.1% TFA over the courseof 30 min);

UV detection (λ=276 nm);

MS (MALDI-TOF): m/z obtained=3739.5 ([MH]+ calculated forC₁₅₆H₂₄₄N₄₅O₄₈S₇=3739.5).

1. A peptide C^(α)-amide precursor of a peptide C^(α)-thioester comprising the radical of general formula (I):

wherein X represents a sulfur or selenium atom, R₁ represents a hydrogen atom or a protective group for the sulfur or for the selenium which is compatible with conditions of elongation by Fmoc SPPS, m represents an integer equal to 0 or 1, one of R₂, R₃, R₄, R₅, R₆ or R₇ represents the radical —B—C-D in which: D represents a hydrogen atom or a solid support suitable for solid phase peptide synthesis (SPPS), C is absent or represents an arm that can be used for SPPS, B represents a divalent radical comprising a heteroatom, the others of said R₂, R₃, R₄, R₅, R₆ or R₇ which do not represent —B—C-D, then represent, independently of one another, a hydrogen atom, or an alkyl radical having from 1 to 5 carbon atoms, or a phenyl radical (—C₆H₅), on the condition that at most two of said radicals R₂, R₃, R₄, R₅, R₆ or R₇ represent at the same time a phenyl, R₈ and R₉ represent, independently of one another, an alkyl radical having from 1 to 10 carbon atoms, or a hydrogen atom, n represents an integer ranging from 0 to 4, A representing an aryl or heteroaryl radical chosen from the group comprising the radical of formula:

wherein R₁₀ represents an alkyl radical having from 1 to 10 carbon atoms, R₁₁ and R₁₂ represent, independently of one another, a hydrogen atom, a halogen atom chosen from the group comprising Cl, Br, I and F, a CN radical, an —NO₂ radical, a —CF₃ radical, a phenyl radical (—C₆H₅), a —CONH₂ radical, an R₁₀ radical, an —OR₁₀ radical, an —SR₁₀ radical, an —N(R₁₀)₂ radical, a —COOR₁₀ radical, a —CONHR₁₀ radical or a —CON(R₁₀)₂ radical, R₁₀ being as previously defined, the radical of formula:

wherein at least one of X₁, X₂, X₃, X₄ and X₅ represents a nitrogen (N) atom, a C—OH radical or a C—SH radical, the others of said X₁, X₂, X₃, X₄ or X₅ which do not represent N, C—OH or C—SH, then representing, independently of one another, a C—R₁₁ radical with R₁₁ as previously defined, the radical of formula:

wherein at least one of X₁, X₂, X₃ and Y₄ represents a nitrogen (N) atom, a C—OH radical or a C—SH radical, the others of said X₁, X₂ or X₃ which do not represent N, C—OH or C—SH, then representing, independently of one another, a C—R₁₁ radical with R₁₁ as previously defined, Y₁, Y₂, Y₃ and Y₄ (on condition that Y₄ does not represent C—OH or C—SH) represent, independently of one another, depending on whether they are linked via a single or double bond, a nitrogen (N) atom or an NR₁₀ group with R₁₀ as previously defined, a CH or CH₂ group, a C—R₁₁ or CHR₁₁ or CR₁₁R₁₂ group with R₁₁ and R₁₂ as previously defined, a carbonyl (C═O), an oxygen (O) atom or a sulfur (S) atom, with the condition that at most two of said Y₁, Y₂, Y₃ and Y₄ represent at the same time an oxygen atom or a sulfur atom, one of said Y₁, Y₂, Y₃ or Y₄ possibly being absent, so as to form a 5-membered ring.
 2. The peptide C^(α)-amide as claimed in claim 1, wherein the divalent radical B represents:

wherein E is absent or represents the -(Xaa)_(i)- group in which: i represents an integer ranging from 1 to 20, each Xaa represents, independently of one another, an amino acid residue, and when i is greater than or equal to 2, each of said Xaa is connected to its neighboring Xaa via a peptide bond, or, in the case where m=1, and R₄ or R₅=—B—C-D, then the divalent radical B preferably represents:

wherein: j represents an integer ranging from 0 to 10, and E is as previously defined.
 3. The peptide C^(α)-amide as claimed in claim 1, wherein C represents an arm that can be used for Fmoc SPPS.
 4. The peptide C^(α)-amide as claimed in claim 3, wherein C represents an acid-labile arm chosen from the group comprising a Rink (4-[(2,4-dimethoxyphenyl)methyl]phenoxyacetyl) arm, a Wang (4-alkoxybenzyl) arm, a Sieber (xanthen-3-yloxyalkyl) arm, a PAL (4-2,5-dimethoxyalkoxybenzyl) arm, a 2-chlorotrityl arm, a PAM (phenylacetamidomethyl) arm, a SASRIN (2-methoxy-4-alkoxybenzyl) arm or an MBHA (4-methyl)benzhydryl arm.
 5. The peptide C^(α)-amide as claimed in claim 1 wherein D represents a solid support suitable for Fmoc SPPS and is preferably chosen from the group comprising a resin sold under the name Pega®, a resin sold under the name Chemmatrix™, a polyacrylamide resin, a polystyrene resin or a polystyrenepolyethylene glycol (PEG) mixed resin.
 6. The peptide C^(α)-amide as claimed in claim 1 wherein n is an integer equal to 0, and the radical A has the formula:

wherein X₁ represents C—OH, and each of X₂, X₃, X₄ and X₅ is as defined in claim
 1. 7. The peptide C^(α)-amide as claimed in claim 6, wherein each of X₂, X₃ or X₅ represents CH and X₄ represents C—R₁₁ with R₁₁ as defined in claim
 1. 8. The peptide C^(α)-amide as claimed in claim 1, in the radical (I), wherein n is an integer equal to 0, and the radical A has the formula:

wherein X₁ represents a nitrogen (N) atom and each of said X₂, X₃, X₄ and X₅ is as defined in claim
 1. 9. The peptide C^(α)-amide as claimed in claim 8 wherein each of X₂, X₃ or X₅ represents CH and X₄ represents CH or C—R₁₁ with R₁₁ representing OCH₃ or N(CH₃)₂.
 10. The peptide C^(α)-amide as claimed in claim 1 wherein n is an integer equal to 0, and the radical A has the formula:

in which X₁ or Y₄ represents C—OH or N, the other said X₁ or Y₄ which does not represent C—OH or N, and also each of said X₂, X₃, Y₁, Y₂ and Y₃ are as defined in claim
 1. 11. The peptide C^(α)-amide as claimed in claim 1 wherein n is an integer equal to 0, and the radical A has the formula:

wherein X₁ or X₂ represents C—OH or N, the other of said X₁ or X₂ which does not represent C—OH or N and also each of said X₃, Y₁, Y₂, Y₃ and Y₄ are as defined in claim
 1. 12. The peptide C^(α)-amide as claimed in claim 1 wherein: m represents 0 or 1, each of said R₃, R₄, R₅, R₆ or R₇ represents H, R₂ represents —B—C-D in which B represents:

and C, D and E are as defined in claim
 1. 13. The peptide C^(α)-amide as claimed in claim 1 wherein m is equal to 0 and X represents a sulfur (S) atom.
 14. The peptide C^(α)-amide as claimed in claim 1 wherein R₁ is a protective group for sulfur chosen from the group comprising trityl (Trt), methyltrityl (Mtt), methoxytrityl (Mmt), xanthenyl (Xan), tri-methoxybenzyl (Tmob), acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), benzamidomethyl (Bam), allyloxycarbonylaminomethyl (Allocam), phthalimidomethyl (Pim), 2-(trimethylsilyl)ethyl, t-butyl (tBu), 2,4,6-trimethylbenzyl, quinolinylmethyl (Qm), diphenyl-4-pyridylmethyl, 1-adamantyl, benzyloxymethyl (BOM), 2-tetrahydropyranyl (Thp), benzylthiomethyl, ethylsulfenyl (SEt), t-butylsulfenyl (StBu), phenylsulfenyl (SPh) and 2,4-dinitrophenyl.
 15. The peptide C^(α)-amide as claimed in claim 1 having the general formula (II):

wherein: Peptide1 represents the R₁₈-(Xaa)_(k)- group in which: k is an integer ranging from 1 to 100 Xaa represents, independently of one another, an amino acid residue originating from an amino acid of formula H-Xaa-OH, and when k is greater than or equal to 2, each of said Xaa is connected to its neighboring Xaa via a peptide bond, R₁₈ is a hydrogen atom or a substituent of the N-terminal end included in the Xaa residue, X, m, n, A and R₁ to R₉ being as defined in claim
 1. 16. A compound having the general formula (Ia):

wherein: X, R₁, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as defined in claim 1 and R₂₀ represents a hydrogen or R₁₄, wherein R₁₄═R₁₃ and R₁₃ represents a protective group for the amine function, or R₁₄ represents a protected aminoacyl residue of formula R₁₃—Xaa- with Xaa representing an amino acid residue originating from an amino acid of formula H-Xaa-OH.
 17. A process for preparing a peptide C^(α)-amide of formula (II) as defined in claim 15 by Fmoc SPPS comprising a step of elongation of the compound (Ia) having the general formula (Ia):

wherein: X, R₁, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as defined in claim 1 and R₂₀ represents a hydrogen or R₁₄, wherein R₁₄═R₁₃ and R₁₃ represents a protective group for the amine function, or R₁₄ represents a protected aminoacyl residue of formula R₁₃—Xaa- with Xaa representing an amino acid residue originating from an amino acid of formula H-Xaa-OH, said elongation step making it possible to add Peptide1.
 18. A process for preparing a peptide C^(α)-thioester of general formula (IX): Peptide1-S—R′  (IX) in which: R′— represents a radical originating from a thiol of formula R′—SH, and is an alkyl, aryl, arylalkyl, cycloalkyl, heterocycloalkyl or heteroaryl group, it being possible for each of said groups to also comprise one or more conventional substituents chosen from halogen, carboxyl, sulfonate, ammonium, alcohol, ether, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, haloalkyl, arylalkyl, heteroarylalkyl and arylheterocycloalkyl Peptide1 is as defined in claim 15, said process comprising a reaction for thioesterification between the peptide C^(α)-amide of formula (II) as defined in claim 15, in which R₁ is equal to H, and a thiol of formula R′—SH, in order to obtain the peptide C^(α)-thioester of formula (IX) as defined above, and the compound of formula (Ib):

in which X, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as defined in claim
 1. 19. A process for preparing a peptide of general formula (X): Peptide1-Yaa-Peptide2-R₁₅  (X) wherein: Peptide1 is as defined in claim 15, Yaa is an amino acid residue originating from an amino acid of formula H-Yaa-OH chosen from the group comprising a cysteine, a homocysteine, a β-mercaptovaline, a β-mercaptoleucine, a β-mercaptoisoleucine, a β-mercaptophenylalanine, a β-mercaptolysine, a β-mercaptoproline, a γ-mercaptovaline, a γ-mercaptoisoleucine, a γ-mercaptoleucine, a γ-mercaptolysine, a γ-mercaptoproline, or an amino acid substituted on its nitrogen atom N^(α) with a group containing a β- or γ-aminothiol function, said group being chosen from the group comprising:

Peptide2=(Xaa)₁, with 1=an integer ranging from 1 to 60, Xaa represents, independently of one another, an amino acid residue originating from an amino acid of formula H-Xaa-OH, and when 1 is greater than or equal to 2, each of said Xaa is connected to its neighbor Xaa via a peptide bond, R₁₅ represents —OH, —NH₂ or a radical of general formula (I) in which X represents a sulfur atom and R₁ is a protective group for sulfur which is stable with respect to a treatment with TFA and stable under NCL conditions, wherein: a peptide C^(α)-amide of general formula (II) as defined in claim 15, in which R₁═H or a group which is labile under NCL native chemical ligation conditions, is reacted, by means of an NCL reaction, with a peptide possessing an N-terminal β- or γ-aminothiol residue of general formula (XI): H-Yaa-Peptide2-R₁₅  (XI) in which Yaa, R₁₅ and Peptide2 are as defined above, in order to obtain the peptide of general formula (X) as defined above and the compound of formula (Ia) having the general formula (Ia):

wherein: X, R₁, m, R₂, R₃, R₄, R₅, R₆, R₇, n, R₈, R₉ and A are as defined in claim 1 and R₂₀ represents a hydrogen or R₁₄, wherein R₁₄═R₁₃ and R₁₃ represents a protective group for the amine function, or R₁₄ represents a protected aminoacyl residue of formula R₁₃—Xaa- with Xaa representing an amino acid residue originating from an amino acid of formula H-Xaa-OH.
 20. The use of a peptide C^(α)-amide as defined in claim 1 for preparing a peptide C^(α)-thioester.
 21. The use of a peptide C^(α)-amide as defined in claim 1 in an NCL native chemical ligation reaction for preparing a peptide or a protein, in particular of therapeutic interest. 